Fig. 4.

M. pneumoniae lipids activate NLR family pyrin domain containing 3 inflammasome (NLRP3) inflammasome to induce interleukin (IL‐1β) secretion. (a) Bone marrow‐derived macrophages (BMDMs) were cultured in 24‐well plates and treated with a nuclear factor kappa B (NF‐κB) inhibitor Bay 11‐7082 for 40 min, and then stimulated with lipids for 18 h; transcriptional levels of NLRP3 mRNA were detected by quantitative real‐time polymerase chain reaction (PCR). (b) BMDMs were pretreated with anti‐Toll‐like receptor (TLR)‐2 or anti‐TLR‐4 neutralization antibody for 1 h. Later, the cells were stimulated with lipids for 12 h and labelled with H₂DCFDA at 37°C for 30 min. The intracellular fluorescence intensity was measured using an immunofluorescence microscope (×40). (c–e) BMDMs were pretreated with 10 mmol/l N‐acetyl‐l‐cysteine (NAC) for 4 h, and then stimulated with 120 μg/ml lipids for 6 h. (c) The expression of the p10 domain of the active caspase‐1 was measured by Western blot; the formation of ASC (apoptosis associated speck‐like protein containing a CARD) specks (×100) was captured by fluorescence microscopy (d), the number of ASC specks was counted over 100 cells in each group (e); the levels of IL‐1β in the supernatants were detected by enzyme‐linked immunosorbent assay (ELISA) (f). The results are representative of similar findings from at least three separate experiments. *P < 0·05 denotes significant differences between the compared groups.